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During a survey period of 9 months, diabetic patients attending an outpatient clinic were asked routinely by the nursing staff to produce their home urine/glucose monitoring charts and diet charts. If they could not do so, the importance of bringing these charts was explained and they were instructed to bring their charts at subsequent visits. In addition, the individual responses of 109 consecutive patients were monitored as index cases. The physicians of these 109 patients were asked to grade each patient according to their estimate of the patient's compliance. This subjective rating was correlated with the actual performance of the patients in bringing charts. During the survey the overall number of patients attending the clinic who brought urine/blood charts increased from 31% to 59%. The corresponding figures for diet charts were 6% and 21%. Before intervention, the group rated "high compliance" was more likely than the "low compliance" rated group to bring urine/glucose charts. However, after adjustment for this baseline difference the subsequent performances of the high and low rated groups were comparable. These two groups of patients also performed similarly with respect to diet charts. It is concluded that patients judged subjectively to have poor compliance were able to modify their behaviour similarly to those assessed as having good compliance.

Many different treatment approaches have been advocated for anorexia nervosa, and most clinicians now incorporate some aspects of behaviour modification in refeeding an emaciated patient. Traditional operant programmes have required the patient to spend prolonged periods of time confined to a solitary existence in a bedroom devoid of her normal possessions. The present paper evaluated the rate of weight gain in 68 patients with anorexia nervosa (100 consecutive admissions to a specialised eating disorders unit) using a lenient, flexible approach to refeeding. Despite this more humane approach, similar results were achieved when compared to other more rigid behavioural programmes in that they gained 0.16 kg/day. This finding is discussed in the context of the rigid application of behavioural programmes which can be seen as punitive, undermine the patient's co-operation and destroy any therapeutic relationship which may develop.

The effect of tryptic degradation on structural and functional properties of the membrane-bound acetylcholine receptor from Torpedo californica has been investigated. Under conditions of proteolysis which resulted in extensive degradation of receptor subunits, the membrane preparations retained their full capability of mediating agonist-induced cation flux as measured in rapid kinetic experiments. Low concentrations on trypsin also cleaved receptor dimers to monomers, and this effect was paralleled by degradation of the Mr 65 000 subunits which are known to contain sulfhydryl group(s) involved in receptor dimerization through an interchain disulfide bond(s). This conversion to monomers occurred at lower trypsin concentrations when the enzyme was added to the outside of the vesicles compared with the effects observed when the enzyme was present inside the vesicles. Similarly Mr 43 000 protein consistently found in preparations of the membrane-bound acetylcholine receptor, which can readily be removed without apparent effect on receptor function, displayed greater susceptibility to proteolysis when trypsin was added to the exterior medium rather than inside the vesicles. The results emphasize the full functionality of the monomeric form of the acetylcholine receptor comprised of four polypeptides.

Few objective data are available concerning the psychological profiles and attitudes of sperm donors in artificial insemination programs. We studied 30 consecutive new volunteers and 45 established sperm donors using the Cattell 16PF personality profile and an attitudinal survey concerning motives for donation, attitudes toward uses of sperm, desire for knowledge of outcomes, and attitudes toward disclosures of identifying and nonidentifying information between the parties to artificial insemination. Donors differed from the population norms in 7 of 16 first-order and 1 of 4 second-order personality factors, and this difference appeared to reflect their self-selection as donors. The predominant motive for donation was altruism, but secondary motives were also common. Financial motivation was very low in this population with few students. Donors approved of all current and most hypothetical uses of sperm but were not in favor of disclosures of information to other participants in the program. Restriction of disclosures to nonidentifying details was more acceptable. Unmarried donors were more often motivated for nonaltruistic reasons and were more in favor of disclosures. Attitudes of sperm donors were mostly stable over at least 3 years, but an increasing minority with time envisaged circumstances that might lead to their withdrawal from the program.

Six groups of ten women each in active labor at term had epidural catheters placed in the usual manner and received a 3 mL test dose of 2% lidocaine with epinephrine. Groups 1-6 received, respectively, 5, 10, 20, 30, 40 and 50 micrograms of sufentanil diluted to 10 mL with normal saline. Significantly effective analgesia was provided at all sufentanil doses studied, with pain scores decreasing from 8.1 +/- 0.2 at baseline to 2.9 +/- 0.3 at 10 minutes and 1.1 +/- 0.2 at 30 minutes (mean +/- SEM, average for all groups). The duration of analgesia showed a significant (p less than 0.05) relation to sufentanil dose, increasing from 79.1 +/- 11.3 minutes (5-micrograms group) to 137.8 +/- 17.2 minutes (50-micrograms group). There were no serious maternal side effects, although ten patients developed pruritus, four became dizzy, two experienced mild sedation, and one had transient hypotension. No neonatal side effects occurred. Maternal serum sufentanil levels remained below the sensitivity of the assay, or 0.1 ng/ml.

The voltage dependence of binding of the calcium channel antagonist, (+)-[3H]PN200-110, to rat brain synaptosomes and the effects of dihydropyridines on 45Ca2+ uptake have been investigated. Under nondepolarizing conditions (+)-[3H]PN200-110 binds to a single class of sites with a Kd of 0.07 nM and a binding capacity of 182 fmol/mg of protein. When the synaptosomal membrane potential was dissipated either by osmotic lysis of the synaptosomes or by depolarization induced by raising the external K+ concentration, there was a decrease in affinity (approximately 7-fold) with no change in the number of sites. The effects of calcium channel ligands on 45Ca2+ uptake by synaptosomes have been measured as a function of external potassium concentration, i.e., membrane potential. Depolarization led to a rapid influx of 45Ca2+ whose magnitude was voltage-dependent. Verapamil (100 microM) almost completely inhibited calcium uptake at all potassium concentrations studied. In contrast, the effects of dihydropyridines (2 microM) appear to be voltage-sensitive. At relatively low levels of depolarization (10-25 mM K+) nitrendipine and PN200-110 completely inhibited 45Ca2+ influx, whereas the agonist Bay K8644 slightly potentiated the response. At higher K+ concentrations an additional dihydropyridine-insensitive component of calcium uptake was observed. These results provide evidence for the presence of dihydropyridine-sensitive calcium channels in synaptosomes which may be activated under conditions of partial depolarization.

The educational model which proposed knowledge improvement as a necessary condition for behaviour change (or increased compliance!) is wrong. It ignores the reality that patients fail to comply for many reasons, the least of which is insufficient information. This is not to say that group programmes, or certain types of individual intervention, are not effective in producing change in variables other than factual knowledge, particularly psychosocial factors. There is increasing evidence that formal education programmes are associated with changes in attitude, which may be more a result of the interaction between participants than a response to educational content, and that it is these attitudinal changes which are the precursors to behavioural change. The initial enthusiasm of the medical community for diabetes education as the solution to the major problem of non-compliance has now been replaced by a healthy skepticism. Diabetes education, by itself, has little to offer the patient whose metabolic control is inadequate. The effectiveness of formal education programmes in patients with compromised metabolic control is limited to the few cases where specific information, or a general understanding of diabetes in a newly-diagnosed patient, is lacking. Information deficit is a specific diagnosis with a specific remedy and in many cases the most efficient treatment may be formal education. For the vast majority of patients, however, the information gained from attending traditional diabetes education programmes has very limited implications for behaviour change.

To determine the frequency of hemodynamic and respiratory complications during movement within the hospital, we conducted a prospective study involving 36 critically ill, ventilator-dependent patients who needed procedures done outside the intensive care unit. During the first 20 transports, patients received ventilation through a manual resuscitation bag. Arterial blood gas measurements showed frequent changes from baseline with alterations in PCO2 (greater than 10 torr) or pH (greater than 0.05) occurring on 14 occasions. In a subsequent study, 16 patients received ventilation during transit with the aid of a portable mechanical ventilator. Although 6 patients showed changes in arterial blood gas values, mean changes in PCO2 and pH were significantly less than in the group that received manual ventilatory support. (p less than 0.01). Hemodynamic complications of hypotension and cardiac arrhythmia showed a significant correlation with disturbances in arterial blood gases (p less than 0.05). Although limited by the lack of a control period, this study shows that the transport of critically ill patients may result in severe hemodynamic complications; it also suggests that these complications might be prevented by more careful monitoring of ventilation.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH), folate, dihydrofolate, and the inhibitors trimethoprim and methotrexate bind rapidly and reversibly to both dihydrofolate reductase isoenzymes isolated from Escherichia coli RT500. The coenzyme and substrates appear to bind to only one of the mixture of two forms of the isoenzyme present at equilibrium, while the inhibitors bind to both forms. The proportions of the two forms are different for the two isoenzymes and are pH dependent in each case. The measured association rate constants for substrates and inhibitors lie in the range (1--2) x 10(-7) M-1 s-1 at 25 degrees C but are unlikely to be diffusion controlled. The rate constant for NADPH binding is 2 x 10(6) M-1 s-1. The formation of binary complexes takes place through a multistep mechanism. A minimum of three steps is required to explain the kinetic results. An equilibrium between two or more forms of the enzyme--ligand complex governs the overall dissociation. The stability of this equilibrium is largely responsible for the tighter binding of inhibitors relative to substrate or coenzyme and also for the different binding strengths of inhibitors to the isoenzymes.

Torpedo acetylcholine receptor (AcChR) exists predominantly as dimers, formed by two monomers held together by a disulfide bridge(s). The dimers are easily cleaved to monomers by reducing agents. 2-mercaptoethanesulfonic acid is shown to be a membrane-impermeant reducing agent which cleaves receptor dimers when it is present only on the outside of intact membrane vesicles. There is no increase in the extent of cleavage when 2-mercaptoethanesulfonic acid is also loaded inside the vesicles. Therefore the disulfide bond(s) involved in the dimerization of the Torpedo acetylcholine receptor is (are) formed by cysteine residues which are exposed on the extracellular side of the membrane.

The fluorescent probe 5-(iodoacetamido)salicylic acid has been used to alkylate acetylcholine receptor enriched membrane fragments from Torpedo californica following their reduction with low concentrations of dithiothreitol. This modification did not affect the equilibrium binding of carbamylcholine to the receptor. The fluorescence of bound 5-(iodoacetamido)salicyclic acid was enhanced when the labeled membrane fragments were mixed with carbamylcholine. This increase in fluorescence was abolished by preincubation of the membrane fragments with excess alpha-bungarotoxin and was therefore specific for the acetylcholine receptor. Estimates of dissociation constants obtained from centrifugation experiments with radioactive ligand and from fluorescence titration data were in good agreement, showing that the observed fluorescence enhancement was an accurate reflection of receptor-carbamylcholine complex formation. The kinetics of carbamylcholine binding to labeled membrane fragments have been investigated over a wide range of ligand concentrations by using stopped-flow fluorescence techniques. The kinetic signal was complicated, and four distinct exponential phases were observed. A kinetic mechanism has been proposed to account for this behavior.

A retrospective study was carried out of 116 consecutive patients with anorexia nervosa to ascertain the extent and nature of vegetarianism in this population. Sixty-three (54.3%) patients were found to be avoiding red meat. In only four (6.3%) of these did meat avoidance predate the onset of their anorexia nervosa. Of the remaining 59 patients (best termed pseudovegetarians), 25 (42.4%) patients continued to avoid red meat by the end of treatment. Pseudovegetarianism was associated with a longer duration of anorexia nervosa, a lower weight during the course of their illness, and living away from the parental home. The reintroduction of red meat into the diet was more likely if vegetarianism were of a short duration.

The binding of the GABA receptor agonist [3H]muscimol to membrane preparations from bovine cerebral cortex has been investigated in equilibrium and kinetic experiments. Equilibrium binding curves are biphasic and suggest that [3H]muscimol binds to both high-affinity (Kd approximately 10 nM) and low-affinity (Kd approximately 0.5 microM) sites. Binding to each class of sites is inhibited by GABA and by the specific GABAA receptor antagonist bicuculline. The kinetics of [3H]muscimol binding have been measured by using both manual filtration assays and an automated rapid filtration technique which permits the measurement of ligand dissociation on subsecond time scales. Association and dissociation curves are biphasic at all concentrations of [3H]muscimol studied, even under conditions of low receptor saturation when no significant occupancy of the low-affinity sites would be expected. These results cannot be simply explained by the presence of two populations of binding sites in the membrane preparations but suggest the existence of two forms of the monoliganded receptor. Dissociation constants for these two forms have been estimated to be 16 and 82 nM at 23 degrees C. At higher ligand concentrations, kinetic measurements have suggested that the binding of [3H]muscimol to low-affinity sites is accompanied by a slow conformational change of the receptor-ligand complex.

We have studied alkylation of the membrane-bound acetylcholine receptor (AcChR) from Torpedo californica electric organ by the cholinergic agonist bromo-acetylcholine (BrAcCh). Following reduction of the AcChR with dithiothreitol (DTT) under strictly controlled conditions, a single class of binding sites was covalently labeled by BrAcCh. The extent of alkylation was dependent on the concentration of both DTT and BrAcCh and reached a maximum when a number of sites equivalent to the number of alpha-bungarotoxin (alpha-BTx) binding sites were labeled. The reaction with BrAcCh was completely inhibited by saturating concentrations of alpha-BTx. On the contrary, complete alkylation of the AcChR with [3H]BrAcCh consistently inhibited only approximately 50% of alpha-BTx binding. The effects of DTT reduction and subsequent BrAcCh alkylation on the cation-gating properties of the AcChR were investigated in rapid kinetic experiments. DTT reduction resulted in a slight decrease in the maximum cation flux and a small shift in the effective dissociation constant to higher acetylcholine (AcCh) concentration. The flux response was completely inhibited by maximal alkylation of the membrane vesicles by BrAcCh. A low-affinity binding site for AcCh, which is likely to be important in AcChR activation, has been revealed for T. californica AcChR by studying the effects of cholinergic ligands on the fluorescence of a probe, 4-[(iodoacetoxy)ethylmethylamino]-7-nitro-2,1,3-benzoxadiazole (IANBD), covalently bound to the AcChR protein. Maximal labeling by BrAcCh did not affect the binding of AcCh to the low-affinity binding site, as monitored by changes in the fluorescence of this probe. This low-affinity binding site must therefore be distinct from the site labeled by BrAcCh. The results strongly support the notion that the nicotinic AcChR contains multiple binding sites for cholinergic ligands.

Witkin proposed a maximum likelihood model for the recovery of surface orientation from image texture. We develop two efficient algorithms for solving this shape from texture problem and compare the results of these algorithms with the algorithm described in [1].

The theory behind selection of the ``uniform error'' threshold which equalizes the probability of misclassification in an image containing two classes is presented. It is shown how this threshold can be estimated using local operations. Some examples and possible extensions are considered.

The molecular structure of nicotinic acetylcholine receptors (AcChR) from different peripheral tissues and from different brain areas has been studied. AcChRs from Torpedo and Electrophorus electric organ and from piscine, avian, and mammalian muscle have been shown to be highly conserved proteins composed of four types of homologous subunits (alpha, beta, gamma, and delta) which associate in a stoichiometry alpha 2 beta 1 gamma 1 delta 1 to form a pseudosymmetric pentameric complex molecule. Genealogical analysis suggests that all the subunits of these AcChR derive from a common ancestral gene and that the divergence occurred very early in the evolution of the receptor. This shared ancestry and the very early divergence of the four subunits, as well as their highly conserved structures along the animal evolution, suggests that each of the subunits evolved to perform specific crucial roles in the function of the AcChR complex. In Torpedo the two alpha-subunits present in the AcChR molecule are glycosylated to a different extent. This may be the reason why the binding sites for cholinergic ligands that are located on the alpha-subunits are non-equivalent. The pseudosymmetric pentameric complex has all the properties of a physiological receptor with respect to known parameters leading to postsynaptic depolarization, and therefore contains the molecular structure/structures that constitute a cation selective channel for transport across the membrane. The complex also contains recognition sites for acetylcholine, cholinergic antagonists, polypeptide neurotoxins, small molecule neurotoxins, and local anesthetics. Therefore a multiplicity of receptor-ligand associations are possible. This leads to more complex models of such interactions than previously considered.(ABSTRACT TRUNCATED AT 250 WORDS)

The equilibrium and kinetic properties of agonist binding to the membrane-bound acetylcholine receptor from Torpedo californica have been measured by the fluorescence changes of a probe, 4-[[(iodoacetoxy)ethyl]methylamino]-7-nitro-2,1,3-benzoxadiazole, which was covalently bound to the receptor protein. Dissociation constants for the binding of several agonists have been measured in fluorescence titration experiments, and these are in good agreement with apparent equilibrium constants obtained from the concentration dependence of the cation flux response measured in quantitative in vitro kinetic experiments. These results provide evidence for the existence of a low-affinity binding site for agonists which is likely to be a functionally important site for channel activation. The kinetics of carbamylcholine and acetylcholine binding to this site have been measured in stopped-flow fluorescence experiments. Kinetic traces were recorded over a wide range of agonist concentrations, and all could be fit by a single exponential process whose rate and amplitude increased hyperbolically with the concentration of ligand. The observed signal change has been ascribed to a conformational transition of the receptor-ligand complex, and this occurs on a millisecond time scale at saturating ligand concentrations which is sufficiently fast to suggest a role for this binding site in the process of channel activation. These results indicate that in the Torpedo AcChR activation and desensitization may be parallel processes which are mediated by agonist association with different receptor binding sites.

A fluorescence assay for measuring the functional properties of the GABAA receptor in reconstituted membrane vesicles is described. This assay is based on a method previously described to measure monovalent cation transport mediated by the nicotinic acetylcholine receptor in membranes from Torpedo electric organ [Moore, H.-P.H., & Raftery, M. A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 4509-4513]. The GABAA receptor has been solubilized from bovine brain membranes and reconstituted into phospholipid vesicles. Influx of chloride or iodide into the vesicles has been measured in stopped-flow experiments by monitoring the fluorescence quench of an anion-sensitive fluorophore trapped within the vesicles. Muscimol, a GABAA receptor agonist, stimulated a rapid uptake of either chloride or iodide. Stimulation of chloride influx was dependent on the concentration of muscimol, and the midpoint of the dose-response curve occurred at approximately 0.3 microM. Agonist-stimulated uptake was enhanced by diazepam and blocked by desensitization and by the antagonists bicuculline and picrotoxin. These receptor-mediated effects are shown to be qualitatively similar to measurements of 36Cl- and 125I- efflux using synaptoneurosomes prepared from rat cerebral cortex. The advantages of the fluorescence method in terms of its improved time resolution, sensitivity, and suitability for quantitating GABAA receptor function are discussed.

The kinetics of ligand binding to dihydrofolate reductase from Lactobacillus casei (MTX/R) to form the ternary enzyme-inhibitor-coenzyme complex have been investigated by the stopped-flow fluorescence technique. The fluorescence changes observed when coenzymes or inhibitors bind to the binary complex of the enzyme with the complementary ligand occur in a single fast phase. Under pseudo-first-order conditions the reaction traces could be fitted with precision to a single-exponential decay, and apparent bimolecular rate constants in the range 2 x 10(6) to 3 x 10(7) M-1s-1 have been measured assuming a bimolecular-unimolecular model. The kinetic constants obtained suggest that prior binding of an inhibitor to the enzyme may, to a minor extent, interfere with coenzyme binding but the rates of inhibitor binding seem to be unaffected by the presence of a bound coenzyme. Dissociation rate constants appear to be less than 1 s-1 which suggests that both coenzymes and inhibitors are tightly bound in the ternary complex. An investigation of the effects of pH on the kinetics of ternary complex formation indicated the involvement of ionizable groups in ligand binding, but this shows some ligand dependence. The rates of ligand bindings to form the ternary complex are fairly high, but it is unlikely that these associations are diffusion controlled because their measured activation energies of 7.8-14.5 kcal mol-1 are higher than expected from reactions whose rates are limited by diffusion in aqeous solution.

The effects of local anesthetics and perhydrohistrionicotoxin on the kinetic mechanism of carbamoylcholine binding to the membrane-bound acetylcholine receptor have been studied by stopped-flow methods. Receptor-enriched membrane fragments from Torpedo californica were reduced and then alkylated by 5-(iodoacetamido)salicylic acid, and the agonist binding kinetics were monitored by the fluorescence changes of this bound probe. The alkylation procedures did not alter the ability of the receptor to mediate agonist-induced cation flux. Preincubation of such modified receptor preparations with saturating concentrations of lidocaine, prilocaine, or dimethisoquin did not significantly affect the equilibrium dissociation constant for carbamoylcholine binding. The multiphasic kinetic signal which accompanies the binding of the agonist was, however, much simplified in the presence of local anesthetics, and the observed kinetics could be described by a mechanism in which a single conformational change follows the formation of the initial complex. Perhydrohistrionicotoxin did not act in the same way as the local anesthetics examined since saturating concentrations did not significantly perturb the agonist binding kinetics.